Vacuolar ATPases (V-ATPases;V1VO-ATPases) are large, multi subunit protein complexes found in the endomembrane system of eukaryotic organisms where they function to acidify the interior of subcellular compartments. In polarized cells of higher eukaryotes, the vacuolar ATPase can also function in the plasma membrane in order to pump protons to the outside of the cell. The proton pumping action of the vacuolar ATPase is involved in a large number of intra- and inter cellular processes such as receptor mediated endocytosis, protein trafficking, pH homeostasis, storage of metabolites and neurotransmitter release. Given its widespread nature, it is not surprising that more and more diseases as fundamental as diabetes, cancer, osteoporosis and AIDS are found to be associated with a defective human vacuolar ATPase. We are studying the structure of this important enzyme by electron microscopy, X-ray crystallography and solution nuclear magnetic resonance spectroscopy as well as other biophysical techniques. In the first funding period for this project, we have generated three-dimensional structural models of the mammalian- and yeast vacuolar ATPase. Furthermore, by using difference mapping, immuno labeling and fitting of X-ray crystal structures, we have been able to determine the subunit architecture of the V-ATPase complex. We are now proposing to extend our structural studies and to use a variety of independent techniques to obtain high resolution structural data for the mammalian- and yeast vacuolar ATPase and for the vacuolar like ATPase from Archaea. Furthermore, we propose to test a number of hypotheses regarding the mechanism by which the ATPase driven proton pumping activity of the vacuolar ATPase is regulated in vivo. The specific aims for this competing continuation proposal are: 1) to investigate the structure and function of the peripheral stalk(s), 2) to elucidate the mechanism of activity silencing in the V1-ATPase domain 3) to determine the subunit architecture of the yeast VO domain by electron crystallography. From the high resolution structural data for the vacuolar ATPase will hope to better understand the catalytic mechanism of ATP hydrolysis powered proton pumping and the mechanism of reversible dissociation/reassociation by which the enzyme's activity is regulated in vivo.